Oxygen is vital to most human and animal life. It can, however, give rise to a variety of reactive oxygen species (“ROS”) as part of normal metabolism. Reactive species are produced by the body under normal conditions, and indeed are part of normal metabolism. The body is equipped with a variety of mechanisms which render ROS inactive.
Under normal conditions, the rate of ROS production does not exceed the capacity of the tissue to catabolize them. However, under certain conditions, ROS levels are raised beyond the capacity of these protective mechanisms (e.g., irradiation, environmental factors, iron loading, etc.) or when these mechanisms are faulty (e.g., genetic defects), and the ROS can cause cellular and tissue damage leading to a variety of diseases and even death. Proteins, lipids, and DNA are all substrates for ROS attack. It has been calculated that for every 100 tons of oxygen consumed two tons form reactive oxygen species. For every 1012 oxygen molecules entering a cell each day {fraction (1/100)} damages protein and {fraction (1/200)} damages DNA. It is this damage to DNA, proteins, and lipids that makes the reactive oxygen species so dangerous, especially when the body's natural defenses are compromised.
Increasing evidence suggests that oxidative stress plays an important role in aging. The level of some antioxidant enzymes such as sodium oxide dismutase (SOD) and antioxidants such as uric acid, beta-carotene and vitamin E have a positive correlation with the life-span of species. Namely, the level decreases from human to chimpanzee to mouse (Culter, Free Radicals in Biology, vol. 4: p. 371, 1984). One hypothesis is that cells are damaged by free radicals and the damaged cells cannot function properly. The accumulation of damages to cells leads to aging (Culter, Id.). Another hypothesis is that free radicals cause cells to dysdifferentiate from their proper state of differentiation. This dysdifferentiation of cells leads to aging and all kinds of age-related diseases. (Culter, Id.). In spite of the disagreement on the mechanism of aging by those skilled in the art, it is clear that free radicals cause aging and age-related diseases. Free radicals have been implicated in stroke, ischemia-reperfusion, cardiovascular diseases, carcingogenesis and neurological diseases, including Alzheimer's disease, Parkinson's disease, dementia and Hodgkin's disease.
Complications of atherosclerosis, such as myocardial infarction, stroke and peripheral vascular disease account for half of the deaths in the United States. Arteriosclerosis begins with an injury to the endothelial cells and is associated with the proliferation of muscle cells inside the arteries. In the process of atherosclerosis, blood becomes thick and platelets, oxidized low density lipoprotein (LDL, the major lipid in LDL is cholesterol esters) and other substances begin to adhere to the walls of the arteries causing the formation of plaque. The oxidation of LDL is caused by free radicals. It was first recognized in 1969 (McCully, Amer. J. Pathol. 56:111, 1969), and only recently rediscovered, that high level of plasma homocysteine is associated with an increased rate of death due to coronary artery disease (Nygard et al., N. Engl. J. Med. 24: 337, 1997; Graham et al., JAMA 277:1775, 1997). Homocysteine injures endothelial cells, thereby causing atherosclerosis through a number of mechanisms, including the generation of hydrogen peroxide (H2O2). It has been reported that homocysteine decreased the bioavailability of NO (not its production) and impaired the intracellular antioxidant enzymes, especially the glutathione peroxidases (Upchurch et al., J. Biol. Chem. 272: 17012, 1997). The key event in the process is generation and presence of free radicals. The increase of hydrogen peroxide (H2O2) can be a cause or a result. Homocysteine causes the production free radicals including superoxide (O2•−) which reacts with NO causing its decreased bioavailability and the production of hydroxyl radical (•OH), or undergoes dismutation by SOD to produce hydrogen peroxide (H2O2). Hydrogen peroxide (H2O2) is further converted to the reactive hydroxyl radical (•OH) through the Fenton reaction and the metal-catalyzed Haber-Weiss reaction. The free radicals produced as a result of these reactions will damage the antioxidant enzymes which prevents the detoxification of free radicals. It is clear that scavenging free radicals will prevent the toxic effects of LDL and homocysteine and results in the prevention of atherosclerosis.
Extensive research efforts have been made to counter the damaging effects caused by free radicals which includes the use of antioxidant enzymes and antioxidants. Unfortunately, protein enzymes are too big to penetrate the cell wall and blood brain barrier. Antioxidants alone are not satisfactory for various reasons including the fact that they are consumed by free radicals and, thus, a large quantity is needed.
Several reactive oxygen species exist. Diatomic molecular oxygen (O2) readily reacts to form partially reduced species, which are generally short-lived and highly reactive and include the superoxide anion (O2•−) a free radical), hydrogen peroxide H2O2 and the hydroxyl radicals (•OH).
The ROS are the byproducts of mitochondrial electron transport, various oxygen-utilizing enzyme systems, peroxisomes, and other processes associated with normal aerobic metabolism as well as lipid peroxidation. These damaging byproducts further react with each other or other chemicals to generate more toxic products. For example, hydrogen peroxide H2O2 can be transformed to the highly reactive hydroxyl radical (•OH) through the Fenton reaction and the metal catalyzed Haber-Weiss reaction: 
Superoxide (O2•−) reacts with nitric oxide (NO) to form the toxic peroxynitrite (ONOO−) which further decomposes to release the hydroxyl radical (•OH). 
Human beings have a defense system against toxic byproducts of metabolism including enzymes such as superoxide dismutase (“SOD”), catalases, peroxidases and antioxidants such as vitamins (e.g., vitamin A, beta-carotene, vitamin C and vitamin E), glutathione, uric acid and other phenolic compounds. SOD catalyzes the conversion of superoxide (O2•−) into hydrogen peroxide (H2O2) and oxygen (O2). 
Hydrogen peroxide (H2O2) can be transformed by catalases and peroxidases to oxygen (O2) and water. 
Despite the high efficiency of the defense system, some of these damaging species escape. The escaped reactive oxygen species and their products react with cellular DNA, protein and lipid resulting in DNA damage and peroxidation of membrane lipids. The deleterious results caused by reactive oxygen species are termed oxidative stress which affects normal gene expression, cell differentiation (Culter, Free Radicals in Biology, vol. 4, p.371, 1984; Culter, Ann. New York Acad. Sci. 621: 1, 1991) and leads to cell death. Oxidative stress is now considered to be responsible for many health problems like cardiovascular and neurological diseases, cancer and other aging-related diseases as well as the human aging process.
Receptors to the neuroexcitatory amino acid, glutamate, particularly the N-methyl-D-aspartate (NMDA) subtype of these receptors, play critical roles in the development, function and death of neurons (see, Mc Donald J W et al., Brain Research Reviews, 15: 41-70 (1990) and Choi W, Neuron, 1: 623-34 (1988) incorporated herein by reference). The N-methyl-D-aspartate (NMDA) receptor is a postsynaptic, ionotropic receptor which is responsive to, inter alia, the excitatory amino acids glutamate and glycine and the synthetic compound NMDA, hence the receptor name. The NMDA receptor controls the flow of both divalent (Ca2+) and monovalent (Na+ and K+) ions into the postsynaptic neuronal cell through a receptor associated channel (see, Foster et al., Nature, 329: 395-396 (1987); Mayer et al., Trends in Pharmacol. Sci., 11: 254-260 (1990) incorporated herein by reference).
The NMDA receptor has been implicated during development in specifying neuronal architecture and synaptic connectivity, and may be involved in experience dependent synaptic modifications. In addition, NMDA receptors are also thought to be involved in long-term potentiation, central nervous system (CNS) plasticity, cognitive processes, memory acquisition, retention, and learning. Furthermore, the NMDA receptor has also drawn particular interest since it appears to be involved in a broad spectrum of CNS disorders. For instance, during brain ischemia caused by stroke or traumatic injury, excessive amounts of the excitatory amino acid glutamate are released from damaged or oxygen deprived neurons. This excess glutamate binds to the NMDA receptor which opens the ligand-gated ion channel thereby allowing Ca2+ influx producing a high level of intracellular Ca2+ which activates biochemical cascades resulting in protein, DNA, and membrane degradation leading to cell death. This phenomenon, known as excitotoxicity, is also thought to be responsible for the neurological damage associated with other disorders ranging from hypoglycemia and cardiac arrest to epilepsy. In addition, there are preliminary reports indicating similar involvement in the chronic neurodegeneration of Huntington's, Parkinson's, and Alzheimer's diseases. Activation of the NMDA receptor has been shown to be responsible for post-stroke convulsions, and, in certain models of epilepsy, activation of the NMDA receptor has been shown to be necessary for the generation of seizures. Blockage of the NMDA receptor Ca2+ channel by the animal anesthetic PCP (phencyclidine) produces a psychotic state in humans similar to schizophrenia (reviewed in Johnson et al., Annu. Rev. Pharmacol. Toxicol., 30: 707-750 (1990) incorporated herein by reference). Further, NMDA receptors have also been implicated in certain types of spatial learning, (see, Bliss et al., Nature, 361: 31 (1993), incorporated herein by reference). Interestingly, both the spatial and temporal distribution of NMDA receptors in mammalian nervous systems have been found to vary. Thus, cells may produce NMDA receptors at different times in their life cycles and not all neural cells may utilize the NMDA receptor.
Due to its broad-spectrum of neurological involvement, yet non-universal distribution, investigators have been interested in the identification and development of drugs capable of acting on the NMDA receptor. Drugs that can modulate the NMDA receptor are expected to have enormous therapeutic potential. For instance, U.S. Pat. No. 4,904,681, issued to Cordi et al., and incorporated herein by reference, describes the use of D-cycloserine, which was known to modulate the NMDA receptor, to improve and enhance memory and to treat cognitive deficits linked to a neurological disorder. D-cycloserine is described as a glycine agonist which binds to the strychnine-insensitive glycine receptor.
U.S. Pat. No. 5,061,721, issued to Cordi et al., and incorporated herein by reference, describes the use of a combination of D-cycloserine and D-alanine to treat Alzheimer's disease, age-associated memory impairment, learning deficits, and psychotic disorders, as well as to improve memory or learning in healthy individuals. D-alanine is administered in combination with D-cycloserine to reduce the side effects observed in clinical trials of D-cycloserine, mainly those due to its growth-inhibiting effect on bacteria resulting in depletion of natural intestinal flora. D-Alanine reverses the growth-inhibiting effect of D-cycloserine on bacteria. It is also reported that D-cycloserine actually has partial agonist character.
U.S. Pat. No. 5,086,072, issued to Trullas et al., and incorporated herein by reference, describes the use of 1-aminocyclopropanecarboxylic acid (ACPC), which was known to modulate the NMDA receptor as a partial agonist of the strychnine-insensitive glycine binding site, to treat mood disorders including major depression, bipolar disorder, dysthymia and seasonal effective disorder. It is also therein described that ACPC mimics the actions of clinically effective antidepressants in animal models. In addition, a co-pending U.S. patent application is cited that describes that ACPC and its derivatives may be used to treat neuropharmacological disorders resulting from excessive activation of the NMDA receptor. However, there remains a need in the art for a satisfactory method of modulating NMDA receptor function.
Development of drugs targeting the NMDA receptor, although desirous, has been hindered because the structure of the NMDA receptor has not yet been completely elucidated. It is believed to consist of several protein chains (subunits) embedded in the postsynaptic membrane. The first two subunits determined so far form a large extracellular region which probably contains most of the allosteric binding sites, several transmembrane regions looped and folded to form a pore or channel which is permeable to Ca2+ and a carboxyl terminal region with an as yet unknown function. The opening and closing of the channel is regulated by the binding of various ligands to domains of the protein residing on the extracellular surface and separate from the channel. As such, these ligands are all known as allosteric ligands. The binding of two co-agonist ligands (glycine and glutamate) is thought to effect a conformational change in the overall structure of the protein which is ultimately reflected in the channel opening, partially open, partially closed, or closed. The binding of other allosteric ligands modulates the conformational change caused or effected by glutamate and glycine. It is believed that the channel is in constant motion, alternating between a cation passing (open) and a cation blocking (closed) state. It is not known at present whether the allosteric modulators actually increase the time during which the channel is open to the flow of ions, or whether the modulators increase the frequency of opening. Both effects might be occurring at the same time.
Several compounds are known which are antagonistic to the flow of cations through the NMDA receptor but which do not competitively inhibit the binding of allosteric ligands to any of the known sites. Instead, these compounds bind inside the open cation channel and are generally known as channel blockers. In fact, binding of a tritiated form of one such channel blocker, dizocilpine (i.e., MK-801), is a good measure of the activation of the NMDA receptor complex. When the channel is open, MK-801 may freely pass into the channel and bind to its recognition site in the channel. Conversely, when the channel is closed, MK-801 may not freely pass into the channel and bind. When the channel is partially closed, less MK-801 is able to bind than when the channel is fully open.
Channel blockers such as MK-801 and antagonists are known to protect cells from excitotoxic death but, in their case, the cure may be as undesirable as the death since they block any flux of Ca2+ thereby eliminating any chance of resumed normal activity. Channel blockers and glutamate site antagonists are known to cause hallucinations, high blood pressure, loss of coordination, vacuolation in the brain, learning disability and memory loss. PCP, a typical channel blocker, produces a well characterized schizophrenic state in man.
Other divalent cations such as Mg2+ and Zn2+ can modulate the NMDA receptor. The exact location of the divalent cation binding site(s) is still unclear. Zn2+ appears to be antagonistic to channel opening and appears to bind to an extracellular domain. Mg2+ shows a biphasic activation curve—at low concentrations it is an agonist for NMDA receptor function, and at high concentrations it is a receptor antagonist. It appears to be absolutely necessary for proper receptor functioning and appears to bind at two sites—a voltage dependant binding site for Mg2+ within the channel and another non-voltage dependent binding site on the extracellular domain. These compounds can modulate the NMDA receptor but are not appropriate for long-term therapy.
Furthermore, as recited, glutamate activates the NMDA receptor, increasing levels of intracellular calcium, which leads to activation of proteases, lipases, and other mediators of cell injury. The increasing levels of cellular calcium also results in membrane depolarization and spreading depression, further increasing energy demands and extracellular glutamate. Nitric oxide and other free radicals are generated that damage DNA, proteins, and fatty acids. A variety of neurological and inflammatory disorders may result from the increased levels of cellular calcium.
Thus, there is a need in the art for safe and effective compounds for modulating the Ca2+ flow through the NMDA ion channel for preventing the overproduction of nitric oxide and other free radicals.